Since neurons cannot be collected from a patient with neurodegenerative disease or spinal cord injury resulting from a damage in specific neurons, studies regarding the pathogenic mechanism thereof and the development of a therapeutic method therefor largely depend on an analysis system using model animals. However, in recent years, a case in which a drug that has been effective for a model animal does not show effectiveness in human clinical trials has frequently occurred, and thus, the limitation of studies using animal models has been pointed out. Hence, it has been strongly expected that human-derived neurons, which are produced from human-derived pluripotent stem cells (i.e., embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) as a result of differentiation; see Patent Literatures 1 and 2), will be used as an analysis system, a drug screening system, and further, a cell preparation.
As methods of inducing differentiation of pluripotent stem cells into neural cells, methods of determining cell fates by on extrinsic signals from outside of the cells, such as a method which comprises forming an embryoid body (a cell mass comprising neural progenitor cells) in a serum-free medium and then allowing it to differentiate into neural cells (SFEB method) (Non Patent Literature 1), a method which comprises culturing embryonic stem cells on stromal cells and then allowing the cultured cells to differentiate into neural cells (SDIA method) (Non Patent Literature 2), a method which comprises culturing the pluripotent stem cells with a drug on matrigel (Non Patent Literature 3), and a method using a low molecular weight compound as an alternative to cytokine (Patent Literature 1), have been mainly used so far. However, these methods have been problematic in that it takes a long period of time, such as several months, to obtain neurons of interest, and also in that since the synchrony in differentiation is extremely low, a uniform cell population cannot be obtained. These problems have been pointed out as causes for insufficient reproducibility in data, when the neurons obtained by the above described methods are used in drug screening or cell transplantation. Moreover, there have been reported many cases in which the neurons obtained by these methods do not sufficiently possess intrinsic properties of neurons, or do not reproduce a phenomenon characteristic to the disease (a pathological hallmark) when prepared from pluripotent stem cells derived from a patient with neurodegenerative disease.
Hence, a method of producing neurons from pluripotent stem cells in a short time has been attempted by introducing a forcible expression of certain transcription factors that are scheduled to be expressed in neuronal cell lineage in the process of development. Non Patent Literature 4 discloses a method in which human-derived embryonic stem cells or induced pluripotent stem cells are differentiated into neural progenitor cells by the SFEB method, and then, three types of (motor) neuron lineage-specific transcription factors (Ngn2, Lhx3, and Isl1) are introduced and allowed to express in the neural progenitor cells, so that motor neurons can be obtained 11 days after the introduction. In addition, Non Patent Literature 5 discloses a method in which three types of neuron lineage-specific transcription factors (Ascl1, Brn2, and Mytl1) are introduced and allowed to express in human embryonic stem cells, so that neurons can be obtained approximately 6 days after the introduction.
Thus, a neuron whose differentiation has been induced by the expression of an exogenous gene(s) is referred to as “induced neuron (which is abbreviated as “iN”),” in order to distinguish a neuron whose differentiation has been induced by a signal from the outside of the cells. When the neuron is a motor neuron, the motor neuron is referred to as “induced motor neuron (which is abbreviated as “iMN”)”. As described above, iMN and iN are obtained in an extremely short time, in comparison to the conventional method for producing motor neurons/neurons. However, it cannot be said that the synchrony in differentiation is sufficient, and further, there are harsh opinions regarding the level of reproducibility of the original properties of motor neurons/neurons (in particular, when iMN or iN is derived from a patient with a disease).
Accordingly, a method of producing a motor neuron or a neuron, that sufficiently possesses an intrinsic property of motor neurons/neurons, including a pathological property in patients, from a pluripotent stem cell promptly and synchronically, has been sought-after.